Relation between HPV-16 serology and clinico-pathological data in cervical carcinoma patients: prognostic value of anti-E6 and/or anti-E7 antibodies

 To investigate the clinical significance of the enhanced sensitivity of antibody detection by radio immunoprecipitation assays (RIPA), using in vitro translated HPV-16 E6 and E7 proteins, over synthetic-peptide enzyme-linked immunosorbent assay (ELISA), RIPA for HPV-16 E6 and E7 were performed. The results obtained with E6 and E7 RIPA were related to clinico-pathological data from cervical carcinoma patients positive for HPV type 16 DNA in their primary tumour. The data obtained with E6 and E7 RIPA were then compared to the results obtained using the E7/6-35 synthetic-peptide ELISA. The prevalence of antibodies to E6, E7, E6 and/or E7 and E6 and E7, as determined by RIPA, was significantly higher in cervical cancer patients than in both controls and cervical intraepithelial neoplasia patients. Odds ratios, calculated for cervical carcinoma patients versus controls, ranged from 7.4 to 15.4. Antibodies to E6 and/or E7 were largely restricted to patients with HPV DNA in their primary tumour. Analysis of the relation between prevalence of antibodies to E6 and E7 and clinico-pathological parameters was limited to 85 patients positive for HPV-16 DNA. The strongest relation with clinico-pathological data, such as lesion size, lymph node involvement, and prognosis, was found for E7 synthetic-peptide ELISA, whereas E6 and E7 RIPA did not reach significance. The significance of these findings is discussed. Received: 17 February 1997 / Accepted: 13 March 1997     

人乳头瘤病毒16型E6和E7基因及其突变体转化活性的研究

为筛选出可用于研制HPV治疗性疫苗的HPV16型E6和E7基因突变体,故将HPV16型原型株(德国株)E6和E7基因及其各种突变体分别转染Balb/c3T3细胞,观察转染后的细胞在软琼脂培养中的集落形成能力和在裸鼠体内的成瘤能力.结果表明,单独转染和共转染HPV16野生型E6和E7基因的Balb/c3T3细胞系,在软琼脂中呈集落样生长,并在裸鼠体内成瘤;而转染E6基因突变体mE6(50G)、E7基因的两种突变体mE7-1(24G26G)和mE7-3(24G26G67R)以及共转染mE6和mE7-1的Balb/c3T3细胞,在软琼脂培养中极少形成集落,也不能在裸鼠体内成瘤.提示经结构改造后的HPV16 E6和E7基因已失去了对Balb/c3T3细胞的转化活性,而保留了免疫原性,可用于HPV16相关肿瘤治疗性疫苗的构建.     

Epithelium and fibroblast-like phenotypes derived from HPV16 E6/E7-immortalized human gingival keratinocytes following chronic ethanol treatment

Epithelial-mesenchymal transition (EMT) may be critical for neoplastic progression and its eventual tumorigenicity of epithelia. In this context, we investigated whether EMT and EMT-associated features occurred after chronic ethanol treatment of human gingival keratinocytes immortalized with the E6/E7 oncogenes of human papillomavirus (HPV) type 16. Following a nine-week treatment of cells with 30 mM ethanol in keratinocyte growth medium, they were cultured in normal DMEM with 10% serum. These cell populations were able to proliferate in this medium gradually exhibiting elongated morphology indicating that these cells underwent EMT. Control cells without ethanol treatment did not survive subcultures in DMEM. Upon long-term subcultures of ethanol-treated cells, two phenotypes were obtained exhibiting epithelium-like and spindle-shape fibroblast-like morphology (respectively, termed as EPI and FIB cells), the latter indicating EMT. In comparison to EPI cells, the phenotypic transition to FIB cells was concomitant with a decrease in the expression of keratins, desmoplakins and a complete loss of K14. Moreover, FIB cell transition strongly correlates with an increase in the expression of vimentin and simple epithelial keratin K18. These alterations in FIB cells were associated with the ability of these cells to exhibit anchorage-independent growth, while EPI cells exhibited anchorage-dependent growth. Concerning the transformation stage, FIB cells represent a progressively more advanced transformed phenotype which may reflect an early step during HPV- and ethanol-dependent multi-step carcinogenesis.     

A novel CD4 T-cell epitope described from one of the cervical cancer patients vaccinated with HPV 16 or 18 E7-pulsed dendritic cells

Previously, safety and immunogenicity of human papillomavirus type 16 (HPV16) or 18 E7-pulsed dendritic cells (DC) vaccinations were demonstrated in a dose-escalation Phase I clinical trial which enrolled ten patients diagnosed with stage IB or IIA cervical cancer (nine HPV 16-positive, one HPV 18-positive). The goal of the study was to define the T-cell epitopes of HPV 16 or 18 E7 protein in these patients in order to develop new strategies for treating HPV-associated malignancies. This was accomplished through establishing T-cell lines by stimulating peripheral blood mononuclear cells with autologous mature DC pulsed with the HPV 16 or 18 E7 protein, examining the T-cell responses using ELISPOT assays, and isolating E7-specific T-cell clones based on IFN-γ secretion. Then, the epitope was characterized in terms of its core sequence and the restriction element. Twelve T-cell lines from eight subjects (seven HPV 16-positive, one HPV 18-positive) were evaluated. Positive T-cell responses were demonstrated in four subjects (all HPV 16-positive). All four were positive for the HPV 16 E7 46-70 (EPDRAHYNIVTFCCKCDSTLRLCVQ) region. T-cell clones specific for the E7 47–70 region were isolated from one of the subjects. Further analyses revealed a novel, naturally processed, CD4 T-cell epitope, E7 58–68 (CCKCDSTLRLC), restricted by the HLA-DR17 molecule. This work was supported by the National Institutes of Health (R21CA094507). An erratum to this article can be found at     

Immunization with a poly (lactide co-glycolide) encapsulated plasmid DNA expressing antigenic regions of HPV 16 and 18 results in an increase in the precursor frequency of T cells that respond to epitopes from HPV 16, 18, 6 and 11

A phase II trial was conducted in subjects with human papillomavirus (HPV) associated high-grade cervical dysplasia testing the safety and efficacy of a microparticle encapsulated pDNA vaccine. Amolimogene expresses T cell epitopes from E6 and E7 proteins of HPV types 16 and 18. An analysis was performed on a subset of HLA-A2+ subjects to test whether CD8+ T cells specific to HPV 16, 18, 6 and 11 were increased in response to amolimogene immunization. Of the 21 subjects receiving amolimogene, 11 had elevated CD8+ T cell responses to HPV 16 and/or 18 peptides and seven of these also had increases to corresponding HPV 6 and/or 11 peptides. In addition, T cells primed and expanded in vitro with an HPV 18 peptide demonstrated cross-reactivity to the corresponding HPV 11 peptide. These data demonstrate that treatment with amolimogene elicits T cell responses to HPV 16, 18, 6 and 11.     

Murine T cell lines can change CD25 expression patterns as well as proliferation and cell death patterns in response to interleukin 2

T cell subpopulations were obtained from F12.5 and B245/270D T cell lines during long-term culture. Two altered F12.5 subpopulations proliferated more intensively than the original clone. These two subpopulations of F12.5 constantly expressed CD25 (interleukin 2 receptor α chain) at a high level and exogenously added interleukin 2 (IL-2) enhanced cell death for one of these subpopulations. However, the original clone expressed CD25 only after activation and IL-2 inhibited cell death of the original clone. On the other hand, the altered B245/270D subpopulation lost the antigen-specific proliferation ability. This altered cell line under the stimulation culture did not express CD25 even after activation, although the original line expressed CD25. However, the expression pattern of CD25on the altered cell line at resting state was induced similar to that of the original one. These results suggest that an expression pattern of CD25 can be changed during long-term cultures, accompanied with alteration in response to proliferation and cell death. This revised version was published online in August 2006 with corrections to the Cover Date.